Toward Bifunctional Chelators for Thallium-201 for Use in Nuclear Medicine

Auger electron therapy exploits the cytotoxicity of low-energy electrons emitted during radioactive decay that travel very short distances (typically <1 μm). 201Tl, with a half-life of 73 h, emits ∼37 Auger and other secondary electrons per decay and can be tracked in vivo as its gamma emissions enable SPECT imaging. Despite the useful nuclear properties of 201Tl, satisfactory bifunctional chelators to incorporate it into bioconjugates for molecular targeting have not been developed. H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa are multidentate N- and O-donor chelators that have previously been shown to have high affinity for 111In, 177Lu, and 89Zr. Herein, we report the synthesis and serum stability of [nat/201Tl]Tl3+ complexes with H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa. All ligands quickly and efficiently formed complexes with [201Tl]Tl3+ that gave simple single-peak radiochromatograms and showed greatly improved serum stability compared to DOTA and DTPA. [natTl]Tl-pypa was further characterized using nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and X-ray crystallography, showing evidence of the proton-dependent presence of a nine-coordinate complex and an eight-coordinate complex with a pendant carboxylic acid group. A prostate-specific membrane antigen (PSMA)-targeting bioconjugate of H4pypa was synthesized and radiolabeled. The uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive and PSMA-negative prostate cancer cells was evaluated in vitro and showed evidence of bioreductive release of 201Tl and cellular uptake characteristic of unchelated [201Tl]TlCl. SPECT/CT imaging was used to probe the in vivo biodistribution and stability of [201Tl]Tl-pypa-PSMA. In healthy animals, [201Tl]Tl-pypa-PSMA did not show the myocardial uptake that is characteristic of unchelated 201Tl. In mice bearing DU145 PSMA-positive and PSMA-negative prostate cancer xenografts, the uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive tumors was higher than that in DU145 PSMA-negative tumors but insufficient for useful tumor targeting. We conclude that H4pypa and related ligands represent an advance compared to conventional radiometal chelators such as DOTA and DTPA for Tl3+ chelation but do not resist dissociation for long periods in the biological environment due to vulnerability to reduction of Tl3+ and subsequent release of Tl+. However, this is the first report describing the incorporation of [201Tl]Tl3+ into a chelator–peptide bioconjugate and represents a significant advance in the field of 201Tl-based radiopharmaceuticals. The design of the next generation of chelators must include features to mitigate this susceptibility to bioreduction, which does not arise for other trivalent heavy radiometals.


■ INTRODUCTION
Molecular radionuclide therapy (MRT) involves the delivery of a lethal dose of ionizing radiation emitted by a radionuclide specifically to diseased tissues or tumors. For example, α (such as 225 Ac) and β − (e.g., 177 Lu, 90 Y) emitting radionuclides, attached to antibodies and peptides targeting the prostatespecific membrane antigen (PSMA), have recently shown clinical promise for treating prostate cancer. 1−4 PSMA is expressed on normal prostate cells, but its expression is greatly increased in malignant prostate tissues while remaining low in most other healthy tissues, making it a useful target for MRT. 5 Following treatment with [ 177 Lu]Lu-PSMA-617, 70% of patients experienced a decline in prostate-specific antigen (PSA) levels in the blood. 2 A similar response was observed using [ 225 Ac]Ac-PSMA-617, where patients saw a decline of ≥50% in PSA levels, which is closely associated with better overall survival. 3 Because their typical range in tissues greatly exceeds cellular dimension, β − particles are highly effective at damaging large tumors through the crossfire effect but are much less effective against single tumor cells and small cell clusters. 6,7 In comparison, α particles and radionuclides emitting Auger electrons (AEs) have a high linear energy transfer (LET) (80− 100 and 4−26 keV/μm, respectively), potentially enabling them to target and kill micrometastases and circulating tumor cells. 8,9 α and β − particles travel 40−80 μm and 0.1−10 mm, respectively, which can lead to off-target tissue toxicity to healthy tissues. This can be partially mitigated by choosing radionuclides with emissions that match the tumor size. 9 AEs, on the other hand, travel typically <1 μm, making the likelihood of off-target effects much lower. AE-emitters thus make an exciting group of radionuclides for potentially effective MRT of micrometastases, with few side effects. This is exemplified by a recent report detailing in vitro and preclinical cytotoxic and antitumor effects of AE-emitting [ 125 I]I-DCIBzL as a prostate cancer therapy in preclinical mouse models. 8 161 Tb has also shown therapeutic efficacy through the emission of both beta particles and AEs. In vivo studies using [ 161 Tb]Tb-PSMA-617 showed an improved antitumor effect compared to [ 177 Lu]Lu-PSMA-617 despite the two agents having comparable pharmacokinetics. 10   Bioconjugate Chemistry pubs.acs.org/bc Article positive breast cancer. 11 Radiation doses to the kidney and liver were within radiation toxicity limits, and high tumor accumulation was observed; however, for a therapeutic effect, dose escalation will be required. 11 Michel and co-workers have highlighted the therapeutic potential of antibodies labeled with 67 Ga, as more potency was observed when compared to 111 In and 125 I. 12 Pirovano et al. have developed an 123 I-labeled PARP1 inhibitor ([ 123 I]I-MAPi) utilizing the Auger electron emissions as the basis of a potent radiotherapeutic for use in glioblastoma tumors. 13,14 Thallium-201 ( 201 Tl, t 1/2 = 73 h) has the potential to be a highly effective therapeutic radionuclide in future MRT applications, as it emits 37 Auger and other high LET secondary electrons per decay (c.f. 25 and 12 AEs emitted by 125 I and 161 Tb, respectively). 9,15 Like other AE-emitters, 201 Tl could also facilitate a theranostics and personalized approach with accurate dosimetry as it releases gamma and X-rays, enabling single photon emission computed tomography (SPECT) imaging. Historically, 201 Tl has been used as a SPECT myocardial perfusion imaging agent but has been largely phased out since the introduction of 99m Tc agents like tetrofosmin and sestamibi.
We have recently shown that nontargeted delivery of 201 Tl (in the form of [ 201 Tl]TlCl) shows short-and long-term toxicity in prostate cancer cells. 16 A dramatic decrease in clonogenic survival was achieved at only 0.29 Bq/cell, significantly lower than for other AE-emitting radionuclides such as 67 Ga and 111 In. 17,18 However, [ 201 Tl]Tl + has little intrinsic selectivity for tumors: it accumulates in the myocardium via the Na + /K + ATPase pump. Thus, although it has been a very useful imaging agent for heart function, a targeted approach is required for other in vivo applications. 19 To date, targeted delivery of 201 Tl to cancer cells has been hindered due to the lack of suitable bifunctional chelator chemistry. Despite the high importance of 201 Tl during the early years of nuclear medicine, thallium chelation has been poorly investigated. Previous attempts using proteins conjugated to the most common and broadly useful chelators such as DTPA or DOTA have shown complex instability. 20−22 More recent studies carried out by our group have confirmed that Tl 3+ complexes of EDTA, DTPA, and DOTA, despite forming Tl 3+ complexes with very high association constants, do not possess adequate kinetic stability for MRT, highlighting the continuing need for new thallium chelators that will form kinetically stable complexes. 23 Recently, Orvig and co-workers introduced a range of branched polydentate picolinic acid based chelators for evaluation as chelators for large, high-valent metal ions such as In 3+ , Lu 3+ , Sc 3+ , and Ac 3+ . 24 Figure  1B) show radiochemical yields of >97% after only 10 min of incubation at room temperature (RT). Each chelator was also evaluated with [ 201 Tl]Tl + (i.e., without prior treatment with Iodobeads); no complexation reaction was observed by HPLC in these experiments ( Figure S3).
In Vitro Stability. Each [ 201 Tl]Tl-labeled complex was left standing in an ammonium acetate solution (1 M, pH 5) for 48 h ( Figure S4), and each showed no degradation. However, all complexes showed modest stability when incubated in human serum at 37°C ( Figure 1C). After Figure 2). In complexes of pypa, methylene protons are diastereotopic, with coupling between geminal, diastereotopic methylene protons. In the 1 H COSY spectrum ( Figure  S25) of the pypa complex of Tl 3+ , 12 cross peaks between methylene protons are observed, indicating that at least two chemically distinct [ nat Tl]Tl-pypa complexes are present in the solution that do not interconvert rapidly within the NMR time scale.
X-ray quality single crystals of [Tl(Hpypa)] were obtained by the slow evaporation of equimolar mixtures of TlCl 3 and H 4 pypa solutions in water with the pH adjusted to 2 by the addition of HCl (0.1 M). 30 The crystal structure of [Tl(Hpypa)] is shown in Figure 3, and selected bond lengths can be found in Table 1. Full crystallographic information can be found in Figure S34. The complex has an octacoordinated Tl 3+ in a distorted square antiprismatic geometry, and when grown from a solution at pH 2, one of the carboxylic acid groups is protonated and does not coordinate to Tl 3+ . The Tl 3+ ion is coordinated by eight (N 5 O 3 ) of the nine potential donor atoms of the ligand. The Tl−O bond lengths are between   (7) Å. These are comparable to bond lengths previously reported for Tl 3+ complexes. 31−33 A low symmetry is observed due to the uncoordinated carboxyl group. Numerous attempts were made to grow an X-ray quality crystal at neutral pH or with an alternative counter ion, for example, tetrabutylammonium, but were not fruitful. Under more basic conditions, it is possible that both carboxylate groups coordinate the metal ion, allowing for a higher degree of symmetry. Synthesis of H 4 pypa-PSMA. As a basis for bioconjugate synthesis, an isothiocyanate derivative of H 4 pypa, H 4 pypa-NCS, was synthesized using the method previously described by Li et al. 28,30 To deliver 201 Tl to PSMA-expressing cells, H 4 pypa-NCS must be coupled to the PSMA targeting vector via a linker molecule. Structure−activity relationships (SARs) of several PSMA targeting variants have demonstrated the significant role that linker design can have on the pharmacokinetic profile of a tracer. 34 The linker used here, incorporating a naphthyl group, was chosen due to the desirable characteristics of PSMA-617 in vivo, 34 including the high affinity for PSMA (assisted by the lipophilic linker binding to the hydrophobic PSMA pocket) and fast renal clearance shown by derivative PSMA-617. 34 To prepare the PSMA peptide analogue for coupling to H 4 pypa-NCS, we adapted a previously reported method, as shown in Scheme 1. 35 In brief, L-glutamic acid di-tert-butyl ester was reacted with carbonyldiimidazole (CDI), forming the activated glutamic acid 1. This was then reacted with the cbzprotected L-lysine tert-butyl ester to yield the urea 2. The cbz group was then removed via catalytic hydrogenation, generating the urea derivative 3. Cbz-3-(2-naphthyl)-D-alanine was added via HATU mediated amide coupling in DMF to furnish compound 4 followed by a hydrogenation reaction to remove the cbz group (5). The coupling and cbz deprotection procedures were repeated with cbz-trans-4-(aminomethyl)cyclohexanecarboxylic acid to generate 6 and 7, respectively.
The reaction of a basic solution of H 4 pypa-NCS in chloroform with 7 at ambient temperature led to the formation of conjugate 8 (Scheme 2). The tert-butyl groups of 8 were cleaved using trifluoracetic acid in DCM (1:1) to generate H 4 pypa-PSMA (9), which was purified using reversed-phase HPLC. HR-MS confirmed the formation of the final product 9.
The method previously described for the radiolabeling of  Figure 6A). Mice were then culled, and organs were collected for ex vivo biodistribution ( Figure 6C).  SPECT imaging analysis indicated that radioactivity concentration in DU145 PSMA-positive tumors was consistently higher than in DU145 PSMA-negative tumors and, at  Figure 7C). For PSMA-negative DU145 tumors, 201 Tl radioactivity concentration at 30 min was 2.1 ± 0.2% IA/g and remained steady until 2 h p.i. Biodistribution data 2 h p.i. corroborated SPECT imaging analysis: 201 Tl concentration at 2 h p.i. in DU145 PSMA-positive tumors measured 3.7 ± 2.8% IA/g, and in the PSMA-negative tumors, this 201 Tl radioactivity concentration measured 2.9 ± 1.5% IA/g ( Figure 7B). Imaging and ex vivo biodistribution data further evidenced that [ 201 Tl]Tl-pypa-PSMA is cleared from the blood mainly via a renal pathway, with high levels of radioactivity observed in the kidneys and bladder/urine evident in both imaging and ex vivo biodistribution data. Ex vivo biodistribution data also indicated that the tumor/ blood ratio for PSMA-positive tumors (11.1 ± 1.4) was significantly higher than that for PSMA-negative tumors (3.9 ± 3.0) at 2 h p.i. (p = 0.0385). The tumor/muscle ratio was similarly higher in mice bearing PSMA-positive tumors (ratio of 1.5 ± 0.4) than in mice bearing PSMA-negative tumors (ratio of 0.7 ± 0.2) ( Figure 7E). SPECT image analysis was also used to determine tumor/muscle ratios for [ 201 Tl]Tl-pypa-PSMA. The tumor/muscle ratio for animals bearing PSMAnegative tumors was approximately 1 from 30 min to 2 h p.i. However, the tumor/muscle ratio for animals bearing PSMApositive tumors measured 2.1 ± 0.7 at 30 min and decreased to 1.2 ± 0.4 at 2 h p.i.

■ DISCUSSION
The premise of this work is that to explore the potential of 201 Tl as a therapeutic radionuclide, we need better chelators for thallium, capable of both convenient radiolabeling under mild conditions and resistance to dissociation or transchelation in the biological environment. Chelation of Tl + is likely to be challenging due to the similarity of its aqueous chemical properties to those of group 1 alkali metals. 37 Therefore, in this study, we chose to focus on Tl 3+ .
Established general-purpose chelators widely used for a range of radiometals in nuclear medicine, such as DOTA and DTPA, are excellent chelators for In 3+ (the closest periodic analogue of Tl 3+ ) and indeed form well-defined complexes with Tl 3+ with high affinity. Nevertheless, the DOTA and DTPA complexes of Tl 3+ quickly decompose in serum and cannot be used in Tl 3+ radiopharmaceuticals. 21 No binding constants of either Tl + or Tl 3+ to endogenous serum proteins have been reported in the literature. 38 However, Li et al. have estimated the binding of Tl 3+ to transferrin to have an association constant of 10 22 based on the linear relationship that they have observed between the first hydrolysis constant of the other trivalent group 13 metal ions and their transferrin binding constant. 39 An alternative route to dissociation of Tl 3+ complexes, not available to their In 3+ analogues, is reduction of Tl 3+ to Tl + by reducing agents present in biological media. Because of this unique vulnerability to reduction of Tl 3+ , the analogy to In 3+ and other trivalent heavy metals such as bismuth and lanthanides offers only limited guidance in the design of thallium chelators.  quickly and efficiently under mild conditions and in this respect represent an improvement on DOTA, which required longer reaction times (60 min at room temperature). 23,40 The radiochromatograms of the labeling mixtures each showed single peaks, suggesting the absence of major isomerism or that isomers were rapidly interconvertible (although, at least in the case of the pypa complex, this interpretation is not consistent with 1 H NMR discussed below). On this basis, all four complexes warranted the evaluation of stability in biological media.
The complexes showed no measurable dissociation when incubated in an ammonium acetate buffer or in the presence of transferrin but showed slow decomposition over hours to days in human serum. Although this rate of dissociation is suboptimal, it does not necessarily preclude the use of these chelators in 201 Tl radiopharmaceuticals, and it is significantly better than that reported for EDTA, DTPA, and DOTA: 23 Tl]Tl-neunpa-NH 2 in human serum were comparable after 1 h, with varying degrees of stability after 24 and 48 h. As none of the candidates were ideal with respect to stability, we based the selection of ligands for further evaluation on the ease of incorporation into bioconjugates. 28,30 Additionally, small peptide imaging agents, such as PSMA-617, have very rapid blood clearance (<1 h), so prolonged complex stability (up to 24 or 48 h) is not essential but would be desirable. Thus, for a more detailed evaluation, we selected H 4 pypa, for which a PSMA-targeted peptide bioconjugate has recently been reported. 28 The 1 H NMR spectrum of the [ nat Tl]Tl-pypa complex under mildly basic conditions could be interpreted as consistent with the presence of at least two non-interconverting (on the NMR time scale) species. This is not consistent with the HPLC data reported above for the [ 201 Tl]Tl 3+ complex, which may indicate that the HPLC method used was not capable of resolving multiple isomers/species. An alternative explanation is that in the acidic mobile phase used in HPLC analysis, interconversion between multiple species was rapid because of the dissociation of one or more carboxylate donor groups, which is suppressed under the basic conditions of 1 H NMR but would have allowed the substitution of a carboxylate donor by water or an accessible dissociative mechanism of isomerization.
Crystals of the [Tl(Hpypa)] complex, enabling single crystal XRD analysis, were obtained from an acidic solution. The solid phase structure consists of a complex where one carboxylate group is pendant and protonated, with a Tl 3+ coordination number of eight instead of the potential nine. This suggests that carboxylate group coordination is labile, and while this does not lead to the immediate dissociation or transchelation of Tl 3+ in biological media, it might be expected to increase vulnerability to reduction by decreasing the coordination number and hence reducing electron density on the metal center. This is pertinent to the biological behavior of the complex bioconjugate, discussed below.
The PSMA-pypa conjugate was efficiently radiolabeled with 201 Tl under conditions similar to those used for unconjugated H 4 pypa. The radiolabeled conjugate was biologically evaluated in vitro and in vivo using the prostate cancer cell line DU145 with and without PSMA expression. The in vitro data ( Figure  5) indicate that in the presence of cells, 201 Tl is released from the labeled bioconjugate complex, likely in the form of Tl + : the uptake of radioactivity in cells was initially low but increased with time, and over a period of an hour, the uptake pattern shifted to one that became similar to that of [ 201 Tl]TlCl�that is, it reached levels similar to those typically observed for [ 201 Tl]TlCl. 201 Tl radioactivity uptake was similarly inhibited by potassium ions, was not selective for PSMA-positive cells, and was unaffected by a PSMA-specific blocking agent. This behavior can be interpreted on the basis that during the first few minutes of incubation, before the PSMA-specific binding of the radioconjugate has time to occur to a measurable extent, reducing agents secreted by cells into the medium prior to and after addition of the radioconjugate cause the reduction of [ 201 Tl]Tl 3+ to [ 201 Tl]Tl + and consequent release from the chelator. As this process develops over a period of minutes, the 201 Tl radioactivity behaves biologically as Tl + and is taken up efficiently by cells through the activity of the Na + /K + -ATPase pump, irrespective of PSMA expression.
This interpretation also accounts for the in vivo behavior as observed by SPECT imaging and ex vivo biodistribution.
[ 201 Tl]Tl + shows the characteristic early myocardium uptake expected of a Na + /K + -ATPase substrate and myocardial imaging agent. This behavior is not greatly changed when the [ 201 Tl]Tl + is oxidized to [ 201 Tl]Tl 3+ before administration, consistent with the very rapid reduction upon initial exposure to the biological environment when unprotected by a Tl 3+ chelator. The radiolabeled bioconjugate, on the other hand, shows a greatly reduced early uptake in the myocardium, indicating that the chelator survives and protects the Tl 3+ from reduction and dissociation long enough to allow blood clearance (mainly via the kidney), potentially allowing the opportunity for modest selective uptake in PSMA-expressing tumors, as observed in the in vivo experiments on tumorbearing mice. Although both suppression of myocardial uptake and a degree of PSMA-specific tumor uptake are observed, the tumor uptake is far below that required for effective imaging or treatment and is much less than is commonly observed with other PSMA-based tracers in this tumor model. 36 The results are consistent with the hypothesis that dissociation is promoted by the reduction of the radiometal. This may well be facilitated by the acid-promoted release of a carboxylate donor, as observed in the X-ray crystal structure. The metal is left with reduced electron density and hence greater susceptibility to reduction.

■ CONCLUSIONS
Seeking effective chelators for Tl 3+ , we have evaluated a series of polydentate N, O-ligands that have previously been shown to be effective chelators of other trivalent heavy metal ions often used in nuclear medicine. The findings indicate that the ligands form Tl 3+ complexes more rapidly and efficiently than conventional chelators (DOTA, DTPA) and resist dissociation or transchelation in buffers free of biomolecules or reducing agents. In serum, however, dissociation occurs over several Bioconjugate Chemistry pubs.acs.org/bc Article hours, albeit more slowly than is the case for DOTA and DTPA complexes. 23 With H 4 pypa as an example studied in more detail, it became clear that bioreductive dissociation occurred much more quickly in the presence of living cells than in serum, leading to cellular uptake in vitro that was characteristic of [ 201 Tl]TlCl. In vivo, a [ 201 Tl]Tl-labeled pypa-PSMA conjugate possessed sufficient kinetic stability to show suppression of myocardial uptake and observable but practically inadequate selective delivery to PSMA-positive tumors. We conclude that the class of ligands studied here represents an advance on DOTA and DTPA but is not satisfactory as a basis for thallium-chelating bifunctional chelators. Further design improvement is needed, and this needs to take into account not only simple association/ dissociation constants but also protection against reduction� by maximizing electron density donated to metal by maximizing the coordination number (by building in rigidity and preorganization) and incorporating more strongly electron donating donor groups.  13 C NMR, HSQC, and COSY data were acquired on a Bruker 400 MHz and analyzed using the MestReNova software. Flash chromatography purification was performed on a Biotage Isolera 4 flash chromatography system using Sfar chromatography columns (silica and C18). HPLC was performed on an Agilent 1260 Infinity instrument with UV spectroscopic detection at 254 nm and a Lablogic Flow-Count detector with a Bioscan Inc. B-FC-3200 photomultiplier tube detector and analyzed using the Lablogic Laura software. The mobile phase used for analytical and semipreparative reversedphase HPLC was composed of (A) water with 0.1% TFA and (B) MeCN with 0.1% TFA. LC/MS data were acquired on an Agilent 1200 Series Liquid Chromatograph with UV spectroscopic detection at 254 nm and the same column details as in reversed-phase HPLC, interfaced with an Advion Expression LC/MS mass spectrometer with an electrospray ionization source. The mobile phase used for LC/MS was composed of (A) water with 0.1% formic acid and (B) MeCN with 0.1% formic acid using an Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm). High-resolution electrospray mass spectrometry was carried out by Dr. Lisa Haigh (mass spectrometry service at Imperial College). Crystallographic data were collected using an Agilent Xcalibur PX Ultra A diffractometer, and the structures were refined using the SHELXTL 43 (2 μL) were removed at intervals and analyzed using RP-TLC to assess the stability. In addition to human serum, this process was repeated using an ammonium acetate solution (1 M, pH 5).

■ MATERIALS AND METHODS
Radiolabeling Tissue Culture. DU145 (PSMA-negative) and DU145-PSMA (PSMA-positive) human prostate cancer cells were cultured in an RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/ streptomycin (Sigma-Aldrich, UK) and maintained at 37°C in a humidified atmosphere with 5% CO 2 . 45 PSMA expression was evaluated using FACS, and the results can be found in Figure S1. To study tracer uptake in tumors, SCID/beige mice (male 5−7 weeks old, n = 3 per group) were injected subcutaneously with DU145-PSMA or DU145 cells (4 × 10 6 cells in 100 mL PBS) in the left shoulder. Once tumors had reached 5−10 mm in diameter (4−5 weeks after inoculation), [ 201 Tl]Tl-pypa-PSMA (10.7−24.5 MBq, 20 mmol) was administered via tail vein injection under isoflurane anesthesia. Mice were maintained under continuous anesthesia and imaged by SPECT/CT for up to 2 h post injection. Animals were then euthanized by cervical dislocation. SPECT images were reconstructed using the HiSPECT (Scivis GmbH) reconstruction software package at 0.3 mm isotropic voxel size using standard reconstruction with 35% smoothing and nine iterations. After euthanasia, organs were harvested from the mice, weighed, and gamma counted.

SPECT Scanning and Biodistribution in Healthy and
Image Analysis. Images were analyzed using VivoQuant 2.5 (InviCRO LLC., Boston, USA), enabling the delineation of regions of interest (ROIs) for quantification of radioactivity. ROIs for the tumor and organs (heart, muscle, etc.) were drawn using CT images, and volumes were determined. The total activity in the whole animal (excluding the majority of tail, out of SPECT field of view) at the time of [ 201 Tl] agents' administration was defined as the injected activity (IA), and the percentage of injected activity per cm 3 (% IA/cm 3 ) and amount of radioactivity in tissues (MBq) were determined. A 5 mL syringe with 3 mL of [ 201 Tl]TlCl (40 MBq) was used to calibrate the SPECT/CT and ensure correct co-registration between the SPECT and CT.
Statistical Analysis. Data are reported as average ± standard deviation. Statistical analysis was performed using Graphpad Prism Version 7.0c with unpaired t tests used in uptake and a two-way ANOVA with Sidak's multiple comparisons test used for in vivo studies; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.